Petunia-calibrachoa variety sakpxc020

ABSTRACT

A  petunia - calibrachoa  plant designated SAKPXCO20 is disclosed. Embodiments include the seeds of  petunia - calibrachoa  SAKPXCO20, the plants of  petunia - calibrachoa  SAKPXCO20, to plant parts of  petunia - calibrachoa  SAKPXCO20, and methods for producing a plant produced by crossing  petunia - calibrachoa  SAKPXCO20 with itself or with another variety. Embodiments include methods for producing a plant containing in its genetic material one or more genes or transgenes and the transgenic plants and plant parts produced by those methods. Embodiments also relate to varieties, breeding varieties, plant parts, and cells derived from  petunia - calibrachoa  SAKPXCO20, methods for producing other lines or plant parts derived from  petunia - calibrachoa  SAKPXCO20, and the plants, varieties, and their parts derived from use of those methods. Embodiments further include hybrid seeds, plants, and plant parts produced by crossing  petunia - calibrachoa  SAKPXCO20 with another variety.

BACKGROUND

The embodiments recited herein relate to a novel and distinctpetunia-calibrachoa designated SAKPXCO20, and to the seeds, plant parts,and tissue culture produced by that petunia-calibrachoa variety. Allpublications cited in this application are herein incorporated byreference.

Petunia and calibrachoa are closely related. In the 1990's, severalspecies of petunia were crossed with calibrachoa. The resulting hybridoffspring was named Petchoa.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1 shows the overall plant habit of the plant grown in a pot.

FIG. 2 shows a close-up of the flower.

SUMMARY

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary, not limiting in scope.

According to one embodiment, there is provided a petunia-calibrachoaplant which is valued as breeding line enabling the development ofsuperior ornamental petunia-calibrachoa plants.

Another embodiment discloses a petunia-calibrachoa plant, wherein asample of representative sample of plant tissue of saidpetunia-calibrachoa is deposited with a Budapest Depository.

Another embodiment relates to a plant, or a part thereof, produced bygrowing petunia-calibrachoa SAKPXCO20, wherein the plant part comprisesat least one cell of petunia-calibrachoa SAKPXCO20.

Another embodiment relates to tissue culture produced from protoplastsor cells from the petunia-calibrachoa plants disclosed in the subjectapplication, wherein said cells or protoplasts are produced from a plantpart selected from the group consisting of pollen, ovules, embryos,protoplasts, meristematic cells, callus, pollen, leaves, ovules,anthers, cotyledons, hypocotyl, pistils, roots, root tips, flowers,seeds, petiole, and stems.

Another embodiment relates to a tissue or cell culture of regenerablecells produced from the plant of SAKPXCO20 and a petunia-calibrachoaplant regenerated from the tissue or cell culture of SAKPXCO20.

Another embodiment relates to a method of vegetatively propagating theplant of SAKPXCO20, comprising the steps of: collecting tissue or cellscapable of being propagated from a plant of SAKPXCO20; cultivating saidtissue or cells to obtain proliferated shoots; and rooting saidproliferated shoots to obtain rooted plantlets; or cultivating saidtissue or cells to obtain proliferated shoots, or to obtain plantletsand a plant produced by growing the plantlets or proliferated shoots ofsaid plant.

A further embodiment relates to a method for producing an embryo orseed, wherein the method comprises crossing a SAKPXCO20 plant withanother plant and harvesting the resultant embryo or seed.

A further embodiment relates to a method for developing apetunia-calibrachoa plant in a petunia-calibrachoa plant breedingprogram, comprising applying plant breeding techniques comprisingcrossing, recurrent selection, mutation breeding, wherein said mutationbreeding selects for a mutation that is spontaneous or artificiallyinduced, backcrossing, pedigree breeding, marker enhanced selection,haploid/double haploid production, or transformation to thepetunia-calibrachoa plant of SAKPXCO20, or its parts, whereinapplication of said techniques results in development of apetunia-calibrachoa plant.

A further embodiment relates to a method of introducing a mutation intothe genome of petunia-calibrachoa plant SAKPXCO20, said methodcomprising mutagenesis of the plant, or plant part thereof, ofSAKPXCO20, wherein said mutagenesis is selected from the groupconsisting of temperature, long-term seed storage, tissue cultureconditions, ionizing radiation, chemical mutagens, and targeting inducedlocal lesions in genomes, and wherein the resulting plant comprises atleast one genome mutation and producing plants therefrom.

A further embodiment relates to a method of editing the genome ofpetunia-calibrachoa plant SAKPXCO20, wherein said method is selectedfrom the group comprising zinc finger nucleases, transcriptionactivator-like effector nucleases (TALENs), engineered homingendonucleases/meganucleases, and the clustered regularly interspacedshort palindromic repeat (CRISPR)-associated protein9 (Cas9) system, andplants produced therefrom.

A further embodiment relates to a petunia-calibrachoa seed produced bygrowing SAKPXCO20.

A further embodiment relates to a method of producing apetunia-calibrachoa plant, or part thereof, by growing a seed producedon SAKPXCO20.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION

Petunia-calibrachoa variety SAKPXCO20 disclosed in the presentapplication has shown uniformity and stability, as described in thefollowing section via vegetative cuttings and tissue culture.Petunia-calibrachoa variety SAKPXCO20 disclosed in the presentapplication has been asexually reproduced a sufficient number ofgenerations with careful attention to uniformity of plant type and hasbeen increased with continued observation for uniformity. Additionally,petunia-calibrachoa variety SAKPXCO20 produces viable pollen and iscapable of being used as a parental line in breeding programs.

Origin of SAKPXCO20

SAKPXCO20 comprises a new and distinct variety of petunia-calibrachoa(Petchoa) originating in a greenhouse in Kakegawa, Japan. VarietySAKPXCO20 originated from a hybridization in Kakegawa, Japan in December2011. The female parent was a proprietary petunia line named ‘GY2-1E-6’,which had a yellow flower color and mounding plant habit. The maleparent was a proprietary calibrachoa line named ‘AM9-83A-1B-2A’, whichhad a yellow flower color and a compact plant habit.

In December 2011, approximately 34 seeds were obtained from the initialhybridization. In February 2011, the 34 seeds were sown and the plantswere cultivated in a greenhouse. Resulting plants had flower colors ofyellow and cream both having brown veins with mounding and semi-moundingplant growth habits. In July 2012 the breeder selected a plant from thegroup of plants that exhibited a cream flower color and a mounding plantgrowth habit. The line was given the experimental name ‘K2013-J-229’.

In August 2012, the selection was vegetatively propagated to producerooted cuttings and plants of the selection were cultivated andevaluated in an open field. In November 2012, the breeder observed theselected line to have its distinct characteristics remain stable. InDecember 2012, the selection was propagated again and plants werecultivated. In April 2013, the breeder confirmed that the distinctcharacteristics of the selection were fixed and stable. All breedingwork was conducted at Kakegawa Research station in Kakegawa Japan. Theselection was named SAKPXCO20.

Petunia-calibrachoa SAKPXCO20 is a tender perennial and exhibitsexcellent resistance to rain, heat and drought. SAKPXCO20 will nottolerate temperature below 10° C.

Petunia-calibrachoa variety SAKPXCO20 has the following environmentalconditions for plant growth: The terminal 1.0 to 1.5 inches of anactively growing stem was excised. The vegetative cuttings werepropagated in five to six weeks. The base of the cuttings were dippedfor 1 to 2 seconds in a 1:9 solution of DIP 'N GROW (1 solution: 9water), a root inducing solution, immediately prior to sticking into thecell trays. Cuttings were stuck into plastic cell trays having 98 cells,and containing a moistened peat moss-based growing medium. For the firstweek, the cuttings were misted with water from overhead for 10 secondsevery 30 minutes until sufficient roots were formed.

Rooted cuttings were transplanted and grown in 20 cm diameter plasticpots in a glass greenhouse located in Salinas, Calif. Pots contained apeat moss-based growing medium. Soluble fertilizer containing 20%nitrogen, 10% phosphorus and 20% potassium was applied once a day orevery other day by overhead irrigation. Pots were top-dressed with adry, slow release fertilizer containing 20% nitrogen, 10% phosphorus and18% potassium. The typical average air temperature was 24° C.

Petunia-calibrachoa variety SAKPXCO20 has shown uniformity andstability, as described in the following variety descriptioninformation. Petunia-calibrachoa variety SAKPXCO20 was tested foruniformity and stability a sufficient number of generations with carefulattention to uniformity of plant type and has been increased withcontinued observation for uniformity.

Petunia-calibrachoa variety SAKPXCO20 has the following morphologic andother characteristics based primarily on data collected in Salinas,Calif. Plants were approximately three months old and evaluated inJanuary 2017. Color references are to The R.H.S. Colour Chart of TheRoyal Horticultural Society of London (R.H.S.), 4^(th) edition. Anatomiclabels are from The Cambridge Illustrated Glossary of Botanical Terms,by M. Hickey and C. King, Cambridge University.

TABLE 1 VARIETY DESCRIPTION INFORMATION (COMPRISED OF TABLES 1A AND 1B)TABLE 1A Characteristic SAKPXC020 Form Annual or Tender Perennial HabitSemi-mounding Height 14.5 cm from soil line to top of foliage Spread52.0 cm Time to produce a rooted cutting 4 weeks Time to bloom fromasexual propagation 8 to 10 weeks Stem description Dull in appearanceand circular in cross-section Number of branches About 4 main; manysecondary and tertiary branches Stem diameter 4.0 mm on main stems Stemlength 16.0 cm to 21.0 cm Stem color RHS 144B (Yellow-Green) Stemanthocyanin RHS N79A (Purple) Stem internode length 6.0 mm Stempubescence and color Heavy, RHS N155A (White) Leaf arrangement AlternateLeaf shape Elliptic Leaf apex Obtuse Leaf base Attenuate Leaf marginEntire Leaf attachment Sessile Leaf texture (both surfaces) Dull, waxy,and slightly sticky Leaf pubescence and color Moderate, RHS N155A(White) Leaf venation Pinnate Leaf venation color Upper: RHS 147C(Yellow-Green) Lower: RHS 145B (Yellow-green) Leaf length 5.8 cm Leafwidth 2.0 cm Leaf color Upper: RHS 137A (Green) Lower: Closest to RHS147B (Yellow-green) Leaf fragrance Absent Petiole Absent Flowering habitand type Indeterminate, solitary Total number of flowers at time ofevaluation Approximately 25 Flowering requirements Will flower so longas day length is greater than 12 hours and temperature exceeds 13° C.Duration of flower life 5 days Flower shape Funnel shaped with fivefissures and an abrupt acute petal time at the midvein Flower depth 4.2cm Flower diameter 6.2 cm Flower fragrance Absent Corolla arrangementComposed of 5 petals, fused Flower bud shape Ovate Flower bud length 4.0cm Flower bud diameter 7.0 mm Flower bud surface texture Dull, sticky,with heavy pubescence colored RHS N155A (White) Flower bud color Closestto RHS 2C (Yellow) with RHS N92A (Violet-blue) venation that is heaviestat the midpoint and slight RHS 145C (Yellow-green) at tip Pedunclelength 9.0 mm Peduncle diameter 1.5 mm Peduncle pubescence and colorHeavy pubescence colored RHS N155A (White) Peduncle color RHS 144B(Yellow-Green) with very slight RHS N79A (Purple) Calyx descriptionComposed of 5 sepals, fused below the middle Sepal shape EllipticalSepal attachment Sessile Sepal apex Obtuse Sepal base Attenuate Sepalmargin Entire Sepal length 2.6 cm Sepal width 4.0 mm Sepal color Upper:Closest to RHS 137A (Green) Lower: Closest to but lighter than RHS 146B(Yellow-green) Petal shape Obcordate Petal length 2.2 cm Petal width 3.3cm Petal apex Abruptly acute Petal margin Entire Petal texture (bothsurfaces) Glabrous Petal color, upper surface Closest to RHS 4D (Yellow)with RHS 11B (Yellow) and RHS 166C (Greyed-orange) venation and slightRHS 166A (Greyed-orange) at mid- vein Petal color, lower surface Closestto RHS 4D (Yellow) with very slight RHS N79A (Purple) venation andslight RHS 146D (Yellow-green) and RHS N79A (Purple) at mid-vein Corollatube color, inner surface RHS 7A (Yellow) with very heavy RHS 200A(Brown) venation Corolla tube color, outer surface Closest to RHS 4D(Yellow) with RHS 146D (Yellow-green) at mid- vein and RHS N77A (Purple)venation Flower tube pubescence Inner: Absent Outer: Moderate Stamendescription and number 5, free, 1.7 cm to 2.1 cm in length Filamentcolor Closest to RHS 145C (Yellow-Green) Anther color Closest to 9D(Yellow) Pollen color Closest to RHS 9D (Yellow) Ovary descriptionSuperior Pistil description 1 (per inflorescence); 1.9 cm in lengthStigma color RHS 144B (Yellow-Green) Style color and length Closest toRHS 150C (Yellow- Green); 1.7 cm Fruit/seed set No seed productionviewed TABLE 1B Disease and Insect Resistance SAKPXC020 Botrytis cinereaSusceptible Powdery mildew Susceptible Various stem and root rotsSusceptible Tobacco Mosaic Virus Susceptible Impatiens Necrotic SpottedVirus Susceptible Aphids Susceptible to infestation LeafminerSusceptible to infestation Whitefly Susceptible to infestationLepitopdera Susceptible to infestation

SAKPXCO20 is a new and unique variety of intergenericpetunia-calibrachoa owing to its light-yellow flower color andsemi-mounding plant growth habit. SAKPXCO20 is most similar to thecommercial petunia-calibrachoa variety ‘SAKPXC017’ (U.S. Pat. No.28,612), commercially known as ‘SuperCal® Light Yellow; however, thereare differences as described in the table below.

TABLE 2 COMPARISON WITH SIMILAR VARIETY Characteristic SAKPXC020‘SAKPXC017’ Petal color, Closest to RHS 4D Closest to RHS 11D uppersurface (Yellow) with RHS 11B (Yellow) with RHS 1A (Yellow) and RHS 166C(Green-Yellow) veins and (Greyed-Orange) venation RHS 12B (Yellow) eye.and slight RHS 166A (Greyed-Orange) at mid- vein Petal color, Closest toRHS 4D RHS 11D (Yellow) with lower surface (Yellow) with very slight RHS144B (Yellow-Green) RHS N79A (Purple) midveins. venation and slight RHS146D (Yellow-Green) and RHS N79A (Purple) at mid- vein Plant growthSemi-mounding Mounding habit

SAKPXCO20 differs from it's parental lines as described in the tablebelow.

TABLE 3 COMPARISON WITH PARENTAL LINES Male Parent ‘AM9-83A- FemaleParent Characteristic SAKPXC020 1B-2A’ ‘GY2-1E-6’ Flower color LightYellow with Yellow Yellow brown vein Plant growth habit Semi-moundingCompact Mounding

Further Embodiments

Breeding with Petunia-Calibrachoa Variety SAKPXCO20

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Promising advanced breeding varieties are thoroughly tested and comparedto appropriate standards in environments representative of thecommercial target area(s) for three or more years. The best varietiesare candidates for new commercial varieties; those still deficient in afew traits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, are time-consuming and require precise forward planning,efficient use of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardvariety. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of petunia-calibrachoa breeding is to develop new and superiorpetunia-calibrachoa varieties and hybrids. The breeder initially selectsand crosses two or more parental varieties, followed by repeated selfingand selection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selection, selfing, and mutations.

Using Petunia-Calibrachoa Variety SAKPXCO20 to Develop OtherPetunia-Calibrachoa Varieties

Petunia-calibrachoa varieties such as petunia-calibrachoa varietySAKPXCO20 are a source of breeding material that may be used to developnew petunia-calibrachoa varieties. Plant breeding techniques known inthe art and used in a petunia-calibrachoa breeding program include, butare not limited to, recurrent selection, mass selection, bulk selection,mass selection, backcrossing, pedigree breeding, open pollinationbreeding, restriction fragment length polymorphism enhanced selection,genetic marker enhanced selection, making double haploids,transformation, and gene editing. These techniques can be usedsingularly or in combinations. There are many analytical methodsavailable to evaluate a new variety. The oldest and most traditionalmethod of analysis is the observation of phenotypic traits, butgenotypic analysis may also be used.

Additional Breeding Methods

Any plants produced using the SAKPXCO20 plants disclosed in the presentapplication as at least one parent are also an embodiment. These methodsare well-known in the art and some of the more commonly used breedingmethods are described herein. Descriptions of breeding methods can befound in one of several reference books (e.g., Allard, “Principles ofPlant Breeding” (1999); and Vainstein, “Breeding for Ornamentals:Classical and Molecular Approaches,” Kluwer Academic Publishers (2002);Callaway, “Breeding Ornamental Plants,” Timber Press (2000).

Breeding steps that may be used in the petunia-calibrachoa varietySAKPXCO20 plant breeding program can include for example, pedigreebreeding, backcrossing, mutation breeding, and recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example, SSR markers),Gene Editing and the making of double haploids may be utilized.

As used herein, the term “plant” or plant part includes plant cells,plant protoplasts, plant cell tissue cultures from which SAKPXCO20plants can be regenerated, plant calli, plant clumps, and plant cellsthat are intact in plants or parts of plants, such as pollen, ovules,embryos, protoplasts, meristematic cells, callus, pollen, leaves,ovules, anthers, cotyledons, hypocotyl, pistils, roots, root tips,seeds, flowers, petiole, pods, shoot, or stems and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such aspetunia-calibrachoa variety SAKPXCO20 and another petunia-calibrachoavariety having one or more desirable characteristics that is lacking orwhich complements petunia-calibrachoa variety SAKPXCO20. If the twooriginal parents do not provide all the desired characteristics, othersources can be included in the breeding population. In the pedigreemethod, superior plants are selfed and selected in successive filialgenerations. In the succeeding filial generations, the heterozygouscondition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F₁ to F₂; F₂ to F₃; F₃ to F₄; F₄ to F₅; etc.After a sufficient amount of inbreeding, successive filial generationswill serve to increase seed of the developed variety. Preferably, thedeveloped variety comprises homozygous alleles at about 95% or more ofits loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred variety which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., variety) and the desirable trait transferred from thedonor parent. This is also known as single gene conversion and/orbackcross conversion.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcommercial characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. As used herein,progeny refers to the descendants of one or more of the parental linesand includes an F₁ petunia-calibrachoa plant produced from the cross oftwo petunia-calibrachoa plants where at least one plant includes apetunia-calibrachoa plant disclosed herein and progeny further includes,but is not limited to, subsequent F₂, F₃, F₄, F₅, F₆, F₇, F₈, F₉, andF₁₀ generational crosses with the recurrent parental line. For example,a petunia-calibrachoa plant may be crossed with another variety toproduce a first generation progeny plant. The first generation progenyplant may then be backcrossed to one of its parent varieties to create aBC₁ or BC₂. Progeny are selfed and selected so that the newly developedvariety has many of the attributes of the recurrent parent and yetseveral of the desired attributes of the nonrecurrent parent. Thisapproach leverages the value and strengths of the recurrent parent foruse in new petunia-calibrachoa varieties.

Therefore, another embodiment is a method of making a backcrossconversion of petunia-calibrachoa variety SAKPXCO20, comprising thesteps of crossing petunia-calibrachoa variety SAKPXCO20 with a donorplant comprising a desired trait, selecting an F₁ progeny plantcomprising the desired trait, and backcrossing the selected F₁ progenyplant to petunia-calibrachoa variety SAKPXCO20. This method may furthercomprise the step of obtaining a molecular marker profile ofpetunia-calibrachoa variety SAKPXCO20 and using the molecular markerprofile to select for a progeny plant with the desired trait and themolecular marker profile of petunia-calibrachoa variety SAKPXCO20.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Petunia-calibrachoa variety SAKPXCO20 issuitable for use in a recurrent selection program. The method entailsindividual plants cross pollinating with each other to form progeny. Theprogeny are grown and the superior progeny selected by any number ofselection methods, which include individual plant, half-sib progeny,full-sib progeny, and selfed progeny. The selected progeny are crosspollinated with each other to form progeny for another population. Thispopulation is planted and again superior plants are selected to crosspollinate with each other. Recurrent selection is a cyclical process andtherefore can be repeated as many times as desired. The objective ofrecurrent selection is to improve the traits of a population. Theimproved population can then be used as a source of breeding material toobtain new varieties for commercial or breeding use, including theproduction of a synthetic variety. A synthetic variety is the resultantprogeny formed by the intercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or genotype. These selectedseeds are then bulked and used to grow the next generation. Bulkselection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk, andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Also, instead of self-pollination, directed pollinationcould be used as part of the breeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Protoplast Fusion

Also known as somatic fusion, this process can be used with SAKPXC20 tocreate hybrids. The resulting hybrid plants have the chromosomes of eachparent and thus the process is useful for incorporating new traits. Theprotoplast fusion technique is well known in the art; see for exampleHamill J. D., Cocking E. C. (1988) Somatic Hybridization of Plants andits Use in Agriculture. In: Pais M. S. S., Mavituna F., Novais J. M.(eds) Plant Cell Biotechnology. NATO ASI Series (Series H: CellBiology), vol 18.

Mutation Breeding

Mutation breeding is another method of introducing new traits intopetunia-calibrachoa variety SAKPXCO20. Mutations that occurspontaneously or are artificially induced can be useful sources ofvariability for a plant breeder. The goal of artificial mutagenesis isto increase the rate of mutation for a desired characteristic. Mutationrates can be increased by many different means including temperature,long-term seed storage, tissue culture conditions, ionizing radiation,such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons,(product of nuclear fission by uranium 235 in an atomic reactor), Betaradiation (emitted from radioisotopes such as phosphorus 32 or carbon14), or ultraviolet radiation (preferably from 2500 to 2900 nm);chemical mutagens (such as base analogues (5-bromo-uracil)), relatedcompounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylatingagents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines,sulfates, sulfonates such as ethyl methanesulfonate, sulfones,lactones), sodium azide, hydroxylamine, nitrous acid,methylnitrilsourea, or acridines; TILLING (targeting induced locallesions in genomes), where mutation is induced by chemical mutagens andmutagenesis is accompanied by the isolation of chromosomal DNA fromevery mutated plant line or seed and screening of the population of theseed or plants is performed at the DNA level using advanced moleculartechniques. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found inVainstein, “Breeding for Ornamentals: Classical and MolecularApproaches,” Kluwer Academic Publishers (2002); Sikora, Per, et al.,“Mutagenesis as a Tool in Plant Genetics, Functional Genomics, andBreeding” International Journal of Plant Genomics. 2011 (2011); 13pages. In addition, mutations created in other petunia-calibrachoaplants may be used to produce a backcross conversion ofpetunia-calibrachoa that comprises such mutation.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the embodiments areintended to be within the scope of the embodiments.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system, including Petunia. See for example,Zhang, B. et al., “Exploiting the CRISPR/Cas9 System for Targeted GenomeMutagenesis in Petunia” Science Reports, Vol. 6, February 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:237-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476.

Therefore it is another embodiment to use the CRISPR system onpetunia-calibrachoa variety SAKPXCO20 to modify traits and resistancesor tolerances to pests, herbicides, and viruses.

Gene Editing Using TALENs

Transcription activator-like effector nucleases (TALENs) have beensuccessfully used to introduce targeted mutations via repair of doublestranded breaks (DSBs) either through non-homologous end joining (NHEJ),or by homology-directed repair (HDR) and homology-independent repair inthe presence of a donor template. Thus, TALENs are another mechanism fortargeted genome editing using SAKPXCO20. The technique is well known inthe art; see for example Malzahn, Aimee et al. “Plant genome editingwith TALEN and CRISPR” Cell & bioscience vol. 7 21. 24 Apr. 2017.

Therefore it is another embodiment to use the TALENs system onpetunia-calibrachoa variety SAKPXCO20 to modify traits and resistancesor tolerances to pests, herbicides, and viruses.

Other Methods of Genome Editing

In addition to CRISPR and TALENs, two other types of engineerednucleases can be used for genome editing: engineered homingendonucleases/meganucleases (EMNs), and zinc finger nucleases (ZFNs).These methods are well known in the art. See for example, Petilino,Joseph F. “Genome editing in plants via designed zinc finger nucleases”In Vitro Cell Dev Biol Plant. 51(1): pp. 1-8 (2015); and Daboussi,Fayza, et al. “Engineering Meganuclease for Precise Plant GenomeModification” in Advances in New Technology for Targeted Modification ofPlant Genomes. Springer Science+Business. pp 21-38 (2015).

Therefore it is another embodiment to use engineered nucleases onpetunia-calibrachoa variety SAKPXCO20 to modify traits and resistancesor tolerances to pests, herbicides, and viruses.

Introduction of a New Trait or Locus into Petunia-Calibrachoa VarietySAKPXCO20

Petunia-calibrachoa variety SAKPXCO20 represents a new variety intowhich a new locus or trait may be introgressed. Direct transformationand backcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Molecular Techniques Using Petunia-Calibrachoa Variety SAKPXCO20

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions. Traditional plant breeding has principally been the source ofnew germplasm, however, advances in molecular technologies have allowedbreeders to provide varieties with novel and much wanted commercialattributes. Molecular techniques such as transformation are popular inbreeding ornamental plants and well-known in the art. See Vainstein,“Breeding for Ornamentals: Classical and Molecular Approaches,” KluwerAcademic Publishers (2002).

Breeding with Molecular Markers

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing petunia-calibrachoa variety SAKPXCO20. SeeVainstein, “Breeding for Ornamentals: Classical and MolecularApproaches,” Kluwer Academic Publishers (2002).

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See for example, Fletcher, Richard S., et al., “QTL analysis ofroot morphology, flowering time, and yield reveals trade-offs inresponse to drought in Brassica napus” Journal of Experimental Biology.66 (1): 245-256 (2014). QTL markers can also be used during the breedingprocess for the selection of qualitative traits. For example, markersclosely linked to alleles or markers containing sequences within theactual alleles of interest can be used to select plants that contain thealleles of interest during a backcrossing breeding program. The markerscan also be used to select for the genome of the recurrent parent andagainst the genome of the donor parent. Using this procedure canminimize the amount of genome from the donor parent that remains in theselected plants. It can also be used to reduce the number of crossesback to the recurrent parent needed in a backcrossing program. The useof molecular markers in the selection process is often called geneticmarker enhanced selection. Molecular markers may also be used toidentify and exclude certain sources of germplasm as parental varietiesor ancestors of a plant by providing a means of tracking geneticprofiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a petunia-calibrachoa plant for which petunia-calibrachoavariety SAKPXCO20 is a parent can be used to produce double haploidplants. Double haploids are produced by the doubling of a set ofchromosomes (1N) from a heterozygous plant to produce a completelyhomozygous individual. This can be advantageous because the processomits the generations of selfing needed to obtain a homozygous plantfrom a heterozygous source. For example, see, Ferrie, Alison M. R., etal., “Review of Doubled Haploidy Methodologies in Ornamental Species”Propagation of Ornamental Plants. 11(2): pp. 63-77 (2011).

Thus, an embodiment is a process for making a substantially homozygouspetunia-calibrachoa variety SAKPXCO20 progeny plant by producing orobtaining a seed from the cross of petunia-calibrachoa variety SAKPXCO20and another petunia-calibrachoa plant and applying double haploidmethods to the F₁ seed or F₁ plant or to any successive filialgeneration.

In particular, a process of making seed retaining the molecular markerprofile of petunia-calibrachoa variety SAKPXCO20 is contemplated, suchprocess comprising obtaining or producing F₁ seed for whichpetunia-calibrachoa variety SAKPXCO20 is a parent, inducing doubledhaploids to create progeny without the occurrence of meioticsegregation, obtaining the molecular marker profile ofpetunia-calibrachoa variety SAKPXCO20, and selecting progeny that retainthe molecular marker profile of petunia-calibrachoa variety SAKPXCO20.

Expression Vectors for Petunia-Calibrachoa Transformation: Marker Genes

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). Expression vectors includeat least one genetic marker operably linked to a regulatory element (forexample, a promoter) that allows transformed cells containing the markerto be either recovered by negative selection, i.e., inhibiting growth ofcells that do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin.

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used marker genes forscreening presumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

Expression Vectors for Petunia-Calibrachoa Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions. Many types of promoters are well known in theart.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.Many signal sequences are well-known in the art. See, for example,Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., et al., PlantMol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129(1989); Frontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al.,Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al., J Cell. Biol.,108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991); Kalderon, etal., Cell, 39:499-509 (1984); Steifel, et al., Plant Cell, 2:785-793(1990).

Foreign Protein Genes: Transformation

Various promoters, targeting sequences, enhancing sequences, and otherDNA sequences can be inserted into the genome for the purpose ofaltering the expression of genes.

Many techniques for altering gene expression are well-known to one ofskill in the art, including, but not limited to, knock-outs (such as byinsertion of a transposable element such as Mu (Vicki Chandler, TheMaize Handbook, Ch. 118 (Springer-Verlag 1994)) or other geneticelements such as a FRT, Lox, or other site specific integration sites;antisense technology (see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809(1988) and U.S. Pat. Nos. 5,107,065, 5,453,566, and 5,759,829);co-suppression (e.g., Taylor, Plant Cell, 9:1245 (1997); Jorgensen,Trends Biotech., 8(12):340-344 (1990); Flavell, PNAS USA, 91:3490-3496(1994); Finnegan, et al., Bio/Technology, 12:883-888 (1994); Neuhuber,et al., Mol. Gen. Genet., 244:230-241 (1994)); RNA interference (Napoli,et al., Plant Cell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp,Genes Dev., 13:139-141 (1999); Zamore, et al., Cell, 101:25-33 (2000);Montgomery, et al., PNAS USA, 95:15502-15507 (1998)), virus-induced genesilencing (Burton, et al., Plant Cell, 12:691-705 (2000); Baulcombe,Curr. Op. Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes(Haseloff, et al., Nature, 334:585-591 (1988)); hairpin structures(Smith, et al., Nature, 407:319-320 (2000); U.S. Pat. Nos. 6,423,885,7,138,565, 6,753,139, and 7,713,715); MicroRNA (Aukerman & Sakai, PlantCell, 15:2730-2741 (2003)); ribozymes (Steinecke, et al., EMBO J.,11:1525 (1992); Perriman, et al., Antisense Res. Dev., 3:253 (1993));oligonucleotide mediated targeted modification (e.g., U.S. Pat. Nos.6,528,700 and 6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat.Nos. 7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044);transposable elements (e.g. Dubin, M. J., et al., Transposons: ablessing curse, Current opinion in plant biology, Vol: 42, Page: 23-29,2018 and Eric T. Johnson, Jesse B. Owens & Stefan Moisyadi (2016) Vastpotential for using the piggyBac transposon to engineer transgenicplants at specific genomic locations, Bioengineered, 7:1, 3-6) and othermethods or combinations of the above methods known to those of skill inthe art.

The foregoing methods for transformation may be used for producing atransgenic variety. The transgenic variety could then be crossed withanother (non-transformed or transformed) variety in order to produce anew transgenic variety. Alternatively, a genetic trait that has beenengineered into a particular petunia-calibrachoa variety using theforegoing transformation techniques could be moved into another varietyusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite variety into anelite variety, or from a variety containing a foreign gene in its genomeinto a variety or varieties that do not contain that gene. As usedherein, “crossing” can refer to a simple x by y cross or the process ofbackcrossing depending on the context.

Likewise, by means of one embodiment, genes can be expressed intransformed plants. More particularly, plants can be geneticallyengineered to express various phenotypes of interest, including, but notlimited to, genes that confer resistance to pests or disease, genes thatconfer resistance to an herbicide, genes that confer or contribute to avalue-added or desired trait, genes that control male sterility, genesthat create a site for site specific DNA integration, and genes thataffect abiotic stress resistance. Many hundreds if not thousands ofdifferent genes are known and could potentially be introduced into aplant according to the invention. Non-limiting examples of particulargenes and corresponding phenotypes one may choose to introduce into aplant include one or more genes for insect tolerance, such as a Bacillusthuringiensis (Bt.) gene, pest tolerance such as genes for fungaldisease control, herbicide tolerance such as genes conferring glyphosatetolerance, and genes for quality improvements such as, environmental orstress tolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s). For example, structuralgenes would include any gene that confers insect tolerance including butnot limited to a Bacillus insect control protein gene as described in WO99/31248, herein incorporated by reference in its entirety, U.S. Pat.No. 5,689,052, herein incorporated by reference in its entirety, U.S.Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference intheir entirety. In another embodiment, the structural gene can confertolerance to the herbicide glyphosate as conferred by genes including,but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPSgene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety, or glyphosate oxidoreductasegene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporatedby reference in its entirety. Alternatively, the DNA coding sequencescan affect these phenotypes by encoding a non-translatable RNA moleculethat causes the targeted inhibition of expression of an endogenous gene,for example via antisense- or cosuppression-mediated mechanisms (see,for example, Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991). TheRNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineeredto cleave a desired endogenous mRNA product (see for example, Gibson andShillito, Mol. Biotech., 7:125, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of one or more embodiments.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of ornamental plants andpetunia-calibrachoa SAKPXCO20 and regeneration of plants therefrom iswell-known and widely published. For example, reference may be had toValla Rego, Luciana et al., Crop Breeding and Applied Technology. 1(3):283-300 (2001); Komatsuda, T., et al., Crop Sci., 31:333-337 (1991);Stephens, P. A., et al., Theor. Appl. Genet., 82:633-635 (1991);Komatsuda, T., et al., Plant Cell, Tissue and Organ Culture, 28:103-113(1992); Dhir, S., et al., Plant Cell Reports, 11:285-289 (1992); Pandey,P., et al., Japan J. Breed., 42:1-5 (1992); and Shetty, K., et al.,Plant Science, 81:245-251 (1992). Thus, another embodiment is to providecells which upon growth and differentiation produce petunia-calibrachoaplants having the physiological and morphological characteristics ofpetunia-calibrachoa SAKPXCO20 described in the present application.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such asembryos, pollen, flowers, seeds, pods, petioles, leaves, stems, roots,root tips, anthers, pistils, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One or more aspects may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the embodiments is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope. Theforegoing discussion of the embodiments has been presented for purposesof illustration and description. The foregoing is not intended to limitthe embodiments to the form or forms disclosed herein. In the foregoingDetailed Description for example, various features of the embodimentsare grouped together in one or more embodiments for the purpose ofstreamlining the disclosure. This method of disclosure is not to beinterpreted as reflecting an intention that the claimed embodimentsrequire more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment.

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice of one or more embodiments.

DEPOSIT INFORMATION

A plant tissue deposit of the Sakata Seed America, Inc. proprietarypetunia-calibrachoa variety SAKPXCO20 disclosed above and recited in theappended claims is maintained by Sakata Seed, America, Inc. A depositwill be made with the Provasoli-Guillard National Center for MarineAlgae and Microbiota, Bigelow Laboratory for Ocean Sciences, 60 BigelowDrive, East Boothbay, Me. 04544, United States. Access to this depositwill be available during the pendency of this application to personsdetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. 1.14 and 35 U.S.C. § 122. Upon allowance of anyclaims in this application, all restrictions on the availability to thepublic of the variety will be irrevocably removed by affording access toa deposit of the plant tissue deposit of the same variety with theProvasoli-Guillard National Center for Marine Algae and Microbiota,Bigelow Laboratory for Ocean Sciences. The deposit will be maintained inthe depository for a period of 30 years, or 5 years after the lastrequest, or for the effective life of the patent, whichever is longer,and will be replaced if necessary during that period.

What is claimed is:
 1. A plant of petunia-calibrachoa variety SAKPXCO20,wherein a representative sample of plant tissue of said cultivar wasdeposited under Bigelow NCMA No. ______.
 2. A plant, or a plant partthereof produced by growing the plant of claim 1, wherein the plant orplant part comprises at least one cell of petunia-calibrachoa varietySAKPXCO20.
 3. A petunia-calibrachoa plant, or part thereof, having allof the physiological and morphological characteristics of thepetunia-calibrachoa plant of claim
 1. 4. A tissue or cell culture ofregenerable cells produced from the plant of claim
 1. 5. The tissue orcell culture of claim 4, comprising tissues or cells from a plant partselected from the group consisting of leaves, pollen, embryos,cotyledons, hypocotyl, meristematic cells, roots, root tips, pistils,anthers, flowers, and stems.
 6. A petunia-calibrachoa plant regeneratedfrom the tissue or cell culture of claim 5, wherein said plant has allof the morphological and physiological characteristics ofpetunia-calibrachoa SAKPXCO20 listed in Table
 1. 7. A method ofvegetatively propagating the plant of claim 1, comprising the steps of:collecting tissue or cells capable of being propagated from said plant;cultivating said tissue or cells to obtain proliferated shoots; androoting said proliferated shoots to obtain rooted plantlets; orcultivating said tissue or cells to obtain proliferated shoots, or toobtain plantlets.
 8. A petunia-calibrachoa plant produced by growing theplantlets or proliferated shoots of claim
 7. 9. A method for producingan embryo or seed, wherein the method comprises crossing the plant ofclaim 1 with another plant and harvesting the resultant embryo or seed.10. A method of determining the genotype of the petunia-calibrachoaplant of claim 1, wherein said method comprises obtaining a sample ofnucleic acids from said plant and detecting in said nucleic acids aplurality of polymorphisms.
 11. A method of producing apetunia-calibrachoa plant resistant to the group consisting ofherbicides, insecticides, and disease, wherein the method comprisestransforming the petunia-calibrachoa plant of claim 1 with a transgene,and wherein said transgene confers resistance to an herbicide,insecticide, or disease.
 12. An herbicide, insecticide, or diseaseresistant plant produced by the method of claim
 11. 13. A method fordeveloping a petunia-calibrachoa plant in a plant breeding program,comprising applying plant breeding techniques comprising crossing,recurrent selection, mutation breeding, wherein said mutation breedingselects for a mutation that is spontaneous or artificially induced,backcrossing, pedigree breeding, marker enhanced selection,haploid/double haploid production, or transformation to thepetunia-calibrachoa plant of claim 1, or its parts, wherein applicationof said techniques results in development of a petunia-calibrachoaplant.
 14. A method of introducing a mutation into the genome ofpetunia-calibrachoa plant SAKPXCO20, said method comprising mutagenesisof the plant, or plant part thereof, of claim 1, wherein saidmutagenesis is selected from the group consisting of temperature,long-term seed storage, tissue culture conditions, ionizing radiation,chemical mutagens, or targeting induced local lesions in genomes, andwherein the resulting plant comprises at least one genome mutation. 15.A method of editing the genome of petunia-calibrachoa plant SAKPXCO20,said method comprising editing the genome of the plant, or plant partthereof, of claim 1, wherein said method is selected from the groupcomprising zinc finger nucleases, transcription activator-like effectornucleases (TALENs), engineered homing endonucleases/meganucleases, andthe clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein9 (Cas9) system.
 16. A petunia-calibrachoaplant produced by the method of claim
 15. 17. A petunia-calibrachoa seedproduced by growing the plant of claim
 1. 18. A method of producing apetunia-calibrachoa plant, or part thereof, produced by growing the seedof claim 17.